EP0821237A2 - Essai immunologique pour la détection des anticorps anti-B. burgdorferi et méthode serodiagnostic pour Lyme borreliose, agent diagnostique et kits de tests pour la mise en oeuvre des procédés - Google Patents
Essai immunologique pour la détection des anticorps anti-B. burgdorferi et méthode serodiagnostic pour Lyme borreliose, agent diagnostique et kits de tests pour la mise en oeuvre des procédés Download PDFInfo
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- EP0821237A2 EP0821237A2 EP97112255A EP97112255A EP0821237A2 EP 0821237 A2 EP0821237 A2 EP 0821237A2 EP 97112255 A EP97112255 A EP 97112255A EP 97112255 A EP97112255 A EP 97112255A EP 0821237 A2 EP0821237 A2 EP 0821237A2
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- antigens
- diagnostic agent
- ospc
- afzelii
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/20—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Spirochaetales (O), e.g. Treponema, Leptospira
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
- C07K16/1207—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Spirochaetales (O), e.g. Treponema, Leptospira
Definitions
- the invention relates to an immunoassay for the detection of anti- B . burgdorferi antibodies in a sample.
- the invention relates to diagnostic agents containing, as reagent components, discriminating antigens derived from B. afzelii and / or Ospc antigen from selected RFLP types of Borrelia burgdorferi sl, and to a method for the serodiagnosis of Lyme disease with improved specificity and sensitivity and for differential detection of antibodies against different serotypes of B. Burgdorferi .
- Lyme disease is one of the most common tick-borne infectious diseases in humans. It is a multisystemic disease caused by the spirochete Borrelia burgdorferi .
- B. burgdorferi belongs to the family of spirochetes, ie helical bacteria of 8-30 ⁇ m in length, which consist of an outer shell, the endoflagel cells in the periplasm and the protoplast cylinder.
- the human pathogenic representatives of the spirochetes include relapsing fever borrelia (e.g. B. recurrentis ), the causative agent of syphilis ( Treponema e.g. T. palladium ) and leptospires.
- B. burgdorferi belongs to the family of spirochetes, ie helical bacteria of 8-30 ⁇ m in length, which consist of an outer shell, the endoflagel cells in the periplasm and the protoplast cylinder.
- burgdorferi sensu lato includes the representatives of the species B. burgdorferi sensu stricto, B. garinii, B. afzelii, B. japonica and B. andersonii , with all three of the first-mentioned representatives being widespread in Europe, while B. burgdorferi sensu stricto is predominant in North America.
- Lyme disease can be prevented by administering antibiotics such as Penicillin G, tetracyclines, erythromycin or Cephalosporins. Heals in some cases Lyme disease often spontaneously in the early stages out, however, late manifestations are also not locked out. There is also a clinical cure Antibiotic therapy for late manifestation only in a few To achieve cases. Lyme disease should therefore, if possible be diagnosed early.
- antibiotics such as Penicillin G, tetracyclines, erythromycin or Cephalosporins.
- Lyme disease With the variety of symptoms of Lyme disease is a Evidence of infection through clinical diagnosis in connection with serological methods inevitable.
- Lyme disease With Lyme disease, however, not all three are always Go through stages; smooth transitions and symptom-free Intervals are also possible. There are stadiums too The disease can be skipped in any of the manifest three phases for the first time. This fact leads Again and again to misjudgments, as often accepted becomes that the typical manifestation of Lyme disease, namely the EM must be run through before a later one Manifestation can occur. Because the diverse Appearance of Lyme disease often others Similar forms of disease, it can occur in purely clinical Diagnoses come to misjudgments. This danger increases even more so if the patient's medical history does not Evidence of a previous tick bite.
- the antibody detection is in the serum and at neurological manifestations also from the cerebrospinal fluid carried out. Serological examinations showed Patients with EM predominantly positive IgM findings, in neurological manifestations predominantly positive IgG results. In the late manifestations of ACA and Lyme arthritis the IgG titer is regularly increased and IgM only in Exceptional cases detectable (Wilske et al. 1984. Infection 12: 331-337, Herzer et al. 1986. Zbl. Bakt.Hyg. A 263: 268-274). Exposed persons, such as forest workers, have a hand increasing age often a significantly increased Antibody level.
- the serological findings depend on the stage of Illness, the duration of the symptoms and possibly one antibiotic therapy. So is the antibody detection with previously available test systems for EM only in 20-50%, at neurological manifestations in 50-90% and in ACA and Arthritis possible in 90-100% of cases. Increased antibody titer are not always diagnostically clear as it is both a clinically manifest infection and can act as an epidemic.
- PCR polymerase chain reaction
- the disadvantage of a whole cell lysate ELISA test is that it only indicates whether antibodies react with the antigens used in the ELISA or not. Conformative epitopes are detected on the one hand, but on the other hand it is not possible to distinguish whether this reaction is a specific reaction or a cross reaction. This often leads to false positive results.
- a problem of the specificity of the tests are broad cross-reactions of the B. burgdorferi pathogen to other bacterial pathogens, such as oral spirochetes, other Borrelia and in particular to T. palladium , the pathogen of syphilis. Since the antigens generally consist of lysates of the entire pathogen, antibodies against are also common antigens detected.
- Osp A Osp C
- OspC Some widespread surface proteins with an immunogenic character could be found (Osp A, OspC); however, it was found that these (i) are expressed to different extents by the individual strains and (ii) are also divided into different serotypes within the individual subspecies.
- the lipoprotein OspA can thus be separated into at least 7 different serotypes (Wilske et al., 1993. J. Clin. Microbiol. 31: 340-350).
- the lipoprotein OspC can be separated into at least 35 serotypes (Livey et al., 1995. Mol. Microbiol. 18: 257-269).
- OspC and OspA make it difficult to use these proteins in a diagnostic kit, because in theory all different subtypes would have to be combined. Since sera from Lyme disease patients mostly have antibodies against only one of the three known species, Theisen et al. (1993. J. Clin. Microbiol. 31: 2570-2576) use OspC antigen from all three B. burgdorferi groups ( B. burgdorferi sensu stricto, B. garinii, B.afzelii ) in a serodiagnostic test.
- the proteins P100 (identical to P83 or E90), 41 kD and P39 were described as immunodominant proteins and used in a diagostic. Reactions against P100 can be found in early-stage patients, but corresponding antibodies are usually only formed in the further course of the disease. Antibodies against the 41 kD antigen appear very early, but it turned out that reactions against this antigen itself are not specific for B. burgdorferi , since cross-reactions with other organisms, such as E. coli or Treponemen, have been observed (Luft et al. , 1993. Res. Microbiol. 144: 251-257; Ditton et al., 1992.
- P39 is a protein that is detected especially in the early phase of the disease of antibodies (Ma et al., 1992. J. Clin. Microbiol. 30: 370-376; Aguero-Rosenfeld et al., 1993. J. Clin. Microbiol. 31: 3090-3095). So far, all five proteins mentioned have also been produced recombinantly and tested against various patient and control sera (Wilske et al., 1994. Med. Microbiol. Immunol. 183: 43-59; Fawcett et al., 1993. J. Rheumatol. 20: 734-738).
- EP 0 465 204 discloses antigenic proteins from B. burgdorferi B31 with a molecular weight of 25, 28, 38, 44, 48, 52, 54, 58, 60, 68, 80 and 90 kD (+ -3 kd), which are used in a diagnostic Kit can be used.
- WO 90/04411 describes immunologically active fractions of B. burgdorferi B31 and ACA, which contain antigens with different molecular weights.
- the aim of the present invention is therefore a improved method according to the type mentioned above To provide which is an increased sensitivity and Has specificity, is easy to standardize and Detection of Lyme Disease and how to create one Serodiagnosis also in an early stage of infection as well as Determination of the serotype of the causative infection used can be. Such a test should also be an option provide forecasts for symptoms of the disease to set up.
- the above object is achieved according to the invention by providing a diagnostic agent which, as reacting components, has at least 4, preferably at least 6, most preferably at least 8, of the antigens P870, P412, P402, P365, P315, P310 derived from a membrane fraction of B. afzelii , P280, P251, P230 and P180. It is preferred that the diagnostic agent according to the invention contains all of the above-mentioned antigens. If the diagnostic agent according to the invention does not contain all of the mentioned antigens or if some of the antigens are replaced by others, a sufficiently specific test for Lyme disease may still be present. However, an optimal result can be expected if all of the antigens mentioned are present.
- the antigens contained in a membrane fraction of B. afzelii strain Orth with a molecular weight of 87.0 kD, 41.2 kD, 40.2 kD, 36.5 kD, 31.5 kD, 31.0 kD , 28 kD, 25.1 kD, 23 kD and 18 kD represent discriminating antigens which react specifically with anti- B. burgdorferi antibodies and have no cross-reactivity with other antibodies not specific to Lyme disease. Due to the increased specificity and sensitivity of the antigens contained in the diagnostic agent according to the invention, an improved detection of a seroconversion is possible if Lyme disease is suspected.
- the discriminating antigens were identified by an analysis of the molecular weights of the proteins of a membrane fraction of B. afzelii, which has not yet been described .
- a membrane fraction II from B. afzelii was prepared, which is characterized in particular by the fact that it contains a balanced ratio of different antigens, which prevents overloading of proteins with the same or similar molecular weight as occurs in whole-cell lysates.
- a B. burgdorferi cell preparation consists of several hundred different proteins. In a preparation of a single membrane fraction from B. burgdorferi , a total of 63 different proteins were identified in the context of this invention.
- the proteins of the membrane fraction were separated in a gel electrophoresis system under previously determined, standardized conditions and the molecular weights were determined. Only the improved analysis conditions allowed a clear and reproducible assignment of proteins to a corresponding molecular weight.
- the proteins were identified using a series of monoclonal antibodies, so that it was possible to differentiate between known and unidentified antigens. It was found that three of the B. afzelii proteins are equivalent to known antigens of other B. burgdorferi strains: P870 is equivalent to protein P100 or E90 from the PKo strain; P365 is equivalent to protein 35E and P251 is equivalent to OspC.
- P870 is equivalent to protein P100 or E90 from the PKo strain
- P365 is equivalent to protein 35E
- P251 is equivalent to OspC.
- the diagnostic agent comprises a membrane fraction of B. afzelii, preferably selected from the group of B. afzelii strain Orth and strain H15.
- the B. afzelii strain Orth is deposited with the German Collection for Microorganisms (DMS) under No. 11048 and the strain H15 under No. 11047 in accordance with the Budapest Treaty.
- the diagnostic agent contains, as reagent components, the recombinant antigens P870, P412, P402, P365, P315, P310, P280, P251, P230 and P180, the antigens being derived from antigens of Memoran Fraction II from B. afzelii with a molecular weight of 87.0 kD, 41.2 kD, 40.2 kD, 36.5 kD, 31.5 kD, 31.0 kD, 28 kD, 25.1 kD, 23 kD and 18 kD.
- the advantage of using recombinant antigens is that (i) only specific binding of serum antibodies with these antigens is possible; (ii) it is possible to produce mixtures of homologous antigens of different serotypes or species; (iii) simplify and / or standardize the test; (iv) higher sensitivity is achieved; and (v) the specific antigen is present in the test in sufficient quantities.
- the recombinant antigens can be produced using known genetic engineering methods.
- the methods used for the production of the recombinant antigens include steps such as isolating the antigens of the corresponding molecular weight from the SDS gel, producing monoclonal antibodies, such as those described by Lane et al. (1985. J. Immunol. Method, 81: 223), expression screening of a B. afzelii library with the monoclonal antibodies and identifying and isolating the positive clones expressing the recombinant antigen.
- the antigen can then be isolated and purified from cells which are transfected with the antigen-expressing clone using standard methods.
- the purified antigens can be used as reagent components in the diagnostic agent according to the invention for the detection of Lyme disease.
- Genetic engineering can be carried out with all common prokaryotic and eukaryotic expression systems, such as, for example, with plasmids, which allow expression in host cells such as E. coli, B. subtilis or yeast, in particular in P. pastoris .
- the diagnostic agent according to the invention contains, as reagent components , the purified antigens P870, P412, P402, P365, P315, P310, P280, P251, P230 and P180 derived from a membrane fraction II of B.afzelii .
- the cells of a selected strain of B. afzelii in particular of B. afzelii strain Orth, are lysed according to known methods, the membrane fraction II is separated from the other cell components by selective centrifugation and the proteins by means of a detergent, preferably a non-denaturing detergent, extracted from the membrane.
- the solubilized proteins are then purified using chromatographic methods such as ion exchange chromatography or HPLC.
- the reagent components P870, P412, P402, P365, P315, P310, P280, P251, P230 and P180 are preferably isolated or spatially separated from one another and immobilized on a solid support.
- the diagnostic agent is a membrane fraction of B. afzelii
- the proteins are separated by molecular weight by means of gel electrophoretic methods, such as SDS-PAGE, and then on a solid support, for example on a membrane or a filter, preferably a nitrocellulose membrane. immobilized.
- the proteins a membrane fraction or a mixture which has been purified Contains antigens in a 1-lane SDS gel with a Track width of e.g. 8 cm, separated and on a membrane transferred.
- the transfer of proteins can be done through known methods, such as diffusion technology or Electrical transfer.
- the reagent components of the diagnostic agent bound to a solid support in this way are then used to carry out a Western blot, preferably a modified Western blot in the form of a Surfblot®.
- a Western blot preferably a modified Western blot in the form of a Surfblot®.
- the testing of antibodies against separated and trans-blotted antigens of a selected B. burgdorferi strain is referred to according to the present invention as a vertical analysis.
- the membrane with the bound antigens is either cut into strips, the strips are marked and the individual strips are incubated with a sample, or the membrane is clamped in a Surfblot® apparatus, whereby approximately 30 different samples are brought into contact with the diagnostic agent at the same time can be.
- antigens or in the diagnostic agent genetically engineered recombinant antigens as Reagent components are used, so they are isolated present antigens directly to a solid support, selected from plates, strips, membranes, filters, paper, Film or pearls, immobilized.
- a solid support selected from plates, strips, membranes, filters, paper, Film or pearls, immobilized.
- the reagent components isolated after the purification are mixed in a desired ratio and the mixture, consisting essentially of the purified B. afzelii membrane proteins P870, P412, P402, P365, P315, P310, P280, P251, P230 and P180 , separated by gel electrophoresis according to molecular weight and the antigens bound to a solid support, preferably a membrane or a filter.
- the carrier-bound reagent components are used for a vertical analysis for the detection of B. burgdorferi antibodies.
- the and are isolated purified antigens to produce a line blot used.
- a line blot used.
- they are isolated antigens present on the surface of microtiter plates bound, the individual reagent components preferably be applied in separate depressions of the plate. It can but also a mixture of all antigens on the surface be bound in a plate trough. Testing the samples can then be carried out according to known ELISA test procedures , aliquots of a sample to be tested, for example a human serum for each reagent component given and then a chromogenic substrate reaction is carried out.
- this is carried out the immunoassay of the vertical analysis preferably as a Western Blot or ELISA.
- immunassays such as an enzyme, lumino, Fluoro or radioimmunoassay to perform the be used according to the inventive method.
- the diagnostic according to the invention Agent contains the diagnostic according to the invention Agent as further reagent components at least one OspC Antigen, preferably at least 4, particularly preferred at least 10, most preferably at least 15, im essentially derived from Borrelia RFLP types 1, 2, 5, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 25 and 34.
- the diagnostic agents according to the invention as further Reagent components all of the OspC antigens of the above mentioned Borrelia RFLP types are added. It is also possible that in the diagnostic agent according to the Invention of one or more of these antigens is absent or are replaced by others.
- the diagnostic agent of the present invention is used in particular to carry out an immunoassay for the detection of anti- B. burgdorferi antibodies.
- Another aspect of the present invention comprises a test kit for the detection of B. burgdorferi antibody in a vertical analysis, containing a diagnostic agent of the type described above, reagents and solutions for carrying out detection of an antigen / antibody complex, an evaluation system for establishing a serodiagnosis and instructions, and if necessary a Surfblot® apparatus.
- the test kit according to the invention contains if necessary a Surfblot® or an analog working equipment. Contain reagents and solutions in the test kit according to the invention, buffer such.
- the test kit contains a foil template, the corresponding one Markings on the running position of the discriminatory Has antigens, and an evaluation system.
- the Test kit according to the invention optionally a diagnostic Agent containing at least one OspC as reagent components Antigen, preferably at least 4, particularly preferred at least 10, most preferably at least 15, the essentially of the Borrelia RFLP types 1, 2, 5, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 25 and 34 are derived.
- reagent components can also be be a solid support.
- OspC antigens derived from one representative of the species from B. burgdorferi sensu stricto, B. afzelii and B. garnii, or ideally from all representatives of all known Serovar or RFLP types in a diagnostic test.
- OspC antigen derived from all 35 RFLP types known to date, was tested against a representative panel of human sera, and it was surprisingly found that the majority of patient sera react with at least one of 20 selected RFLP types.
- OspC antigen derived from 20 selected Borrelia RFLP types as reagent components
- antibodies against OspC are known to be formed in an early phase of infection, it has not always been possible to detect anti-OspC antibodies in the immunoassay due to the heterogeneity of OspC.
- OspC-RFLP types According to the invention and their use as reagent components in a diagnostic agent that it is possible to provide a system with high sensitivity and specificity, although a minimum of OspC antigen variants must be used. This is particularly important with regard to the economical production of a highly sensitive diagnostic test, since theoretically all RFLP types should be included.
- a further aspect of the present invention therefore also relates to a diagnostic agent containing, as reagent components, at least 4 OspC antigens, preferably at least 10, particularly preferably at least 15, essentially of the Borrelia RFLP types 1, 2, 5, 7, 8, 9 , 10, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 25 and 34, which is for an immunoassay for the detection of anti- B. burgdorferi antibodies and / or for determining the serotype of the Borrelia pathogen can be used.
- the diagnostic agent according to the invention comprises all OspC antigens of the aforementioned Borrelia RFLP types.
- the diagnostic agent according to the invention is according to one Embodiment of the invention, a membrane fraction, which in essentially of the Borrelia RFLP types 1, 2, 5, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 25 and 34 is derived. Selected representatives of these RFLP types are listed in Table 1. Of course, others can too Representatives of the RFLP types selected according to the invention, the by Livey et al. (1995. Mol. Microbiol. 18: 257-269) be described as the source of the reagent components be used.
- membrane fractions of the selected Borrelia RFLP types are used for the preparation of the diagnostic agent, the membrane fraction is isolated from individual representative strains, the membrane proteins are separated by gel electrophoresis, the proteins are immobilized on a solid support and the immobilized reagent components are used to carry out detection of anti - B. burgdorferi antibodies or used to determine the serotype.
- OspC antigen can also be obtained by isolating and purifying the proteins from Borrelia, as described in EP 0 522 560.
- OspC antigen derived from selected RFLP types from Borrelia, genetically engineered and as a recombinant antigen in diagnostic means used.
- Livey et al. (1995. Mol. Micobiol. 18: 257-269) discloses the amino acid and Nucleotide sequences of OspC-RFLP types for the Production of the different OspC antigen variants using known genetic engineering methods can be used.
- Purified or recombinantly produced Osp-C antigen dissolved in a suitable buffer and on a support immobilized.
- a membrane is used as a solid support, preferably a nitrocellulose membrane used, so it is applied to the membrane in the form of individual Drop of solution (dot blot) or as lines (line blot).
- the diagnostic agent thus produced can then accordingly for carrying out a horizontal western blot analysis be used.
- Another version includes the application of the OspC antigens to a solid Carrier substance, preferably in the form of microtiter plates, the individual reagent components each in one Plate trough can be immobilized. If necessary, too a mixture of some or all of the purified antigens the surface of a well are bound.
- the present invention furthermore comprises the use of a diagnostic agent containing OspC antigens as reagent components, essentially derived from the Borrelia RFLP types 1, 2, 5, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 25 and 34, for the detection of B. burgdorferi antibodies and / or for the determination of the serotype of the Borrelia pathogen.
- a diagnostic agent containing OspC antigens as reagent components, essentially derived from the Borrelia RFLP types 1, 2, 5, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 25 and 34, for the detection of B. burgdorferi antibodies and / or for the determination of the serotype of the Borrelia pathogen.
- the diagnostic agent according to the invention is according to a Another aspect of the invention available in a test kit made of additional reagents and solutions for Carrying out a detection reaction of the formation of a Antigen / antibody complex and an evaluation system for Creating a serodiagnosis and instructions on how to do so.
- antigens derived from a membrane fraction of B.afzelii are essentially used as reagent components in the diagnostic agent derived from Borrelia types 1, 2, 5, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 25 and 34, where the Agents according to the invention can each be used independently of one another in separate immune test systems for the detection of B. burgdorferi antibodies.
- the diagnostic test comprises at least 4 of the aforementioned antigens, preferably all or essentially all of the antigens mentioned.
- the diagnostic agent containing discriminating antigens derived from membrane fraction II from B. afzelii in a vertical analysis preferably a Western surf blot analysis
- the diagnostic agent containing OspC-RFLP antigen variants in a horizontal analysis preferably a line -Blot analysis
- the combination of both analysis systems, vertical and horizontal analysis results in a higher sensitivity and specificity, so that even those samples can be classified as positive or negative for which a clear result was not possible until now.
- the present method offers the advantage that in particular false negative results, but also false positive results that occur as a result of cross reactions, can be excluded.
- other immunoassays known in the art such as an enzyme, lumino, fluoro or radioimmunoassay, can be used both for carrying out the vertical analysis and for the horizontal analysis.
- the present invention therefore also includes aticianstestkit from a diagnosticians means which substantially of a membrane fraction II of B. afzelii derived antigens P870, P412, P402, P365, P315, P310, P280, P251, P230 and P180 contains, for performing a vertical analysis and a diagnostic agent, which as reagent components at least one OspC antigen, preferably at least 4, particularly preferably at least 10, most preferably at least 15 OspC antigens derived from Borrelia RFLP types 1, 2, 5, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 25 and 34 contains, for performing a horizontal analysis for the detection of B. burgdorferi antibodies.
- a diagnostic agent which as reagent components at least one OspC antigen, preferably at least 4, particularly preferably at least 10, most preferably at least 15 OspC antigens derived from Borrelia RFLP types 1, 2, 5, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 25 and 34
- Another aspect of the present invention relates to an immunoassay for the detection of anti- B. burgdorferi antibodies, a diagnostic agent containing, as reagent components, antigens essentially derived from a membrane fraction of B. afzelii P870, P412, P402, P365, P315, P310, P280, P251, P230 and P180, and incubated with a sample suspected of containing B. burgdorferi antibodies under conditions that allow the formation of an antibody / antigen complex and determine the presence of anti - B. burgdorferi antibodies by detection of a reaction product consisting of an antigen / antibody complex, in particular the complex formation of an antibody with one of the reagent components.
- Another aspect of the present invention relates to an immunoassay for the detection of anti- B. burgdorferi antibodies and / or determination of the serotype of the Borrelia pathogen, a diagnostic agent containing, as reagent components, at least 4 OspC antigens derived from the Borrelia RFLP types 1, 2, 5, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 25 and 34, is provided and incubated with a sample that may contains anti- B. burgdorferi antibodies and determine the presence of anti- B. burgdorferi antibodies in the sample by detecting the formation of an antigen / antibody complex, in particular the complex formation of an antibody with an OspC-RFLP-type component.
- Samples that are tested with the method are human or of animal origin, especially blood, plasma, serum, CSF or synovial fluid.
- the test according to the invention is therefore carried out both in the vertical analysis and in the horizontal analysis as a "confirmation assay (confirmation test)", since false negative results are excluded and positive results are confirmed in a further test.
- the sera classified as negative were not further tested in a confirmation assay using previous diagnostic methods.
- the method according to the invention also identified as positive those samples which, despite the late manifestation of the disease, have previously been defined as seronegative.
- the diagnosis of patients with so-called "seronegative Lyme borreliosis” has so far been very difficult and was not possible with conventional serological determination methods.
- the method according to the invention therefore enables a sensitive determination of the seroconversion in patients in an early infection stage as well as in cases in which no clear assignment in seropositive or seronegative was previously possible.
- the method according to the invention makes it possible to detect Borrelia-specific antibodies in samples of the most varied of origins, in particular from different geographical regions, with very high sensitivity and specificity, since the antigen components used in the immunoassay react with almost all known serotypes of Borrelia.
- Seroconversion parameters to determine a positive Seroconversion was carried out according to the present invention as defined as follows:
- Tested samples containing antibodies used in vertical analysis with one of the antigens P870, P412, P402, P365, P315, P310, P280, P251, P230 and P180 from B. afzelii or in horizontal analysis with a selected OspC-RFLP type Antigens forming a complex are classified according to this procedure as responders, i.e. as seroconverted.
- the samples, which are tested with an analysis method, for example a vertical analysis are optionally subjected to a second immunoassay, for example horizontal analysis, to confirm the result, whereby the second test also enables a statement about the serotype of the pathogen.
- the sera in which no antibody-antigen complex formation can be determined with the discriminating antigens of the vertical analysis or in the horizontal analysis, are considered non-responders with respect to the immunassay carried out and are tested with a further immunoassay if necessary to verify the result .
- antibodies to a discriminating antigen of membrane fraction II from B. afzelii may not yet have been formed in the early phase of the infection, it is particularly important to test non-responders in vertical analysis against an antigen which, in particular, antibodies which are in the Early phase of the immune response are recognized.
- the sample of a non-responder, tested with discriminating antigens is therefore only evaluated as seronegative or seropositive in relation to Lyme disease after testing with selected OspC antigens in the horizontal analysis.
- the method is preferably used for Serodiagnosis of Lyme disease and / or to predict the Disease syndrome after Borrelia infection by determination of the syndrome-associated antigen.
- the immunoassay method according to the invention has in particular the advantages that it is (i) easy to manufacture and can be standardized, (ii) by the combination of two Immune test systems gives clear results, with both false negative and false positive are excluded (iii) a large number of samples simultaneously and with reproducible result can be tested and (iv) for the user is easy to handle and evaluate.
- monoclonal antibodies were produced against the antigens identified as discriminatory.
- the present invention therefore also includes monoclonal antibodies against the immunologically active antigens P870, P412, P402, P365, P315, P310, P280, P251, P230 and P180 of a membrane fraction from B. afzelii strain Orth.
- These monoclonal antibodies can be used in an immunoassay in which the antibodies are used as reagent components for the detection of Borrelia antigens in a sample.
- Another use of the monoclonal antibodies is in using them to purify the immunologically active antigens of borrelia.
- the antibodies with known methods on a solid support, for example a column matrix coupled, and the bound antibodies used for affinity chromatography.
- affinity chromatography can identify specific antigens Whole cell lysates from Borrelia or from recombinant Cell cultures expressing the antigen are purified.
- new antigens of membrane fraction II from B. afzelii strain Orth were identified and characterized by their molecular weight.
- the invention therefore also includes immunologically active proteins from B. afzelii strain Orth P870, P412, P402, P365, P315, P310, P280, P230 and P180 with a molecular weight of 87.0 kD, 41.2 kD, 40.2 kD, 36, 5 kD, 31.5 kD, 31.0 kD, 28.0 kD, 23.0 kD and 18.0 kD.
- a serological test system mostly becomes heterologous Responses determined because the one causing an infection Strain is unknown in most cases. Therefore, it is consequently, to choose a test strain that (i) if possible has many potential antigens and (ii) one if possible high reactivity in the reaction with antibodies from Human sera shows.
- membrane fraction II was prepared by two representatives of the species B. burgdorferi ss (B31, HB1), B. afzelii , (Orth, Pko) and B. garinii (K15, W). For this purpose, grown cells were pelleted, the cells disrupted with a Vibrogen® cell mill, the lysate filtered and centrifuged again. The pellet, containing membrane fraction II, was washed with Tris / HCl, resuspended and the amount of protein determined. The membrane fraction II of the three species was analyzed using a modified SDS-PAGE according to Lämmli et al. (1970.
- FIG. 1 shows a typical protein pattern of the Memoran fraction II of the strain Orth. The results of the subsequent counting of the potential antigens (stained with colloidal gold) are shown in FIG. 2. It shows that the representatives of the species B. afzelii and B. garinii have a higher number of potential antigens than the representatives of B. burgdorferi sensu stricto.
- VA Vertical analysis
- the vertical analysis was carried out after a modified method by Towbin et al. (1979. Proc. Natl. Acad. Sci. USA 76: 4350-4354) using Western Surfblot.
- Towbin et al. (1979. Proc. Natl. Acad. Sci. USA 76: 4350-4354) using Western Surfblot.
- To the proteins of a membrane fraction of the strain Orth separated according to Example 1 using a 1-track SDS gel and the antigens were blotted onto an immobilizing membrane. After that, the membrane was 1.5 wide on the right and left The strips are separated and the strips are colored with Auro-Dye®.
- the middle part of the membrane was covered with TBS-Tween 20-BSA blocked and clamped in a surfblot apparatus.
- Fig. 4 shows the results of the vertical analysis of a selection of 37 human sera, tested against the membrane fraction II of the strain Orth.
- On the left are those 49 antigens (in decreasing size in kD) that were reactive with human serum antibodies in the various studies.
- Reuz k r eattentione or n egativ d iskriminante reactions ( KRND reactions) resulted.
- the column next to the molecular weights indicates the names of those antigens that were detectable with known monoclonal antibodies ("E” means "equivalent to” and indicates with the following number the approximate molecular weight of that antigen that is detected by the monoclonal antibody in the homologous strain has been).
- the two other antigens (28.0 kDa, 23.0 kDa) were only assessed as discriminatory by the discriminant analysis, but showed a relevant frequency (32.8-45.3% and 26.2-38.2%) when estimating the frequency of reactions (95% confidence interval estimate). According to the analysis criteria, an antigen only showed one relevant frequency if the lower confidence limit was above a predetermined 20%.
- Two further antigens, 71.3 kDa and 70.4 kDa both are contained in FIG. 5 of the 27 non-redundant antigens) were identified as "negatively discriminatory" by the discriminant analysis.
- the antigens 58.9 kDa (E60), 47.7 kDa, 71.3 kDa and 70.4 kDa are cross-reactive or negatively discriminatory and were therefore excluded.
- Fig. 5 In the two left columns of Fig. 5 are the 29 antigens, which identified as differentiating and non-redundant with their molecular weights and short names cited.
- the three subsequent columns contain Information on each antigen in each of the three sera panels. Table fields marked with "+” indicate those Antigen reactions that (via the redundancy analysis) as differentiated or non-redundant. With Exception of the two antigens 28.0 kDa and 23.0 kDa is too at least one "+" mark is entered for each antigen. The however, neither of the two antigens listed above were found to Redundancy analysis, but by the Discriminant analysis determined.
- the right column contains brief comments on the individual antigen reactions. The gray The highlighted fields indicate those reactions that follow the highest for all analyzes for vertical analysis had analytical relevance.
- afzelii almost all reactions for only 2 RFLP types, namely RFLP type 8 (strain H15) and RFLP type 22 ( Strain E51) was observed. In contrast, species-specific reactions of B. garinii OspC protein evenly distributed and are found in 9 of 12 RFLP types. 6 shows a typical horizontal analysis with OspC variants, tested against various human sera. The order of the Borrelia strains corresponds to that in FIG. 7.
- FIG. 8 shows the reaction pattern of the IgG and IgM response of 37 Patients who were part of a larger study of sera from 190 Patients and 100 blood donors from the ⁇ aland Islands, Finland, was (Serenpanel according to Example 1). The investigation was over carried out to the sensitivity and specificity of the Horizontal western blot analysis in a highly endemic Region to test.
- a positive IgG reaction was marked with "+” and marked a positive IgM response with " ⁇ ”.
- a positive IgG and IgM reaction is marked with " ⁇ ".
- Fig. 8 shows the Evaluation of the Western blot horizontal analysis of the Membrane fraction II from 20 representative strains of the three Main species with corresponding patient sera, which also were used for the vertical analysis according to Example 1. The lower columns of Fig. 8 show the "species specificity" the IgG and IgM response for each individual Serum.
- Assay variant A is used for a general screen for positive reactivity in horizontal analysis.
- Assay variant B can be used to distinguish between the general categories of reaction types in the human immune response. The same criteria apply as described in Example 2b.
- Assay variant C allows the extent of cross-reactivity to be assessed in the test sera and in those sera which show an RFLP-type strain-specific immune response.
- FIGS Figures 4 and 8 show the results of the serum examples from FIGS Figures 4 and 8 shown.
- 33 sera detected as seroconverted will.
- nine sera could only be administered via the Horizontal analysis and two sera exclusively via the Vertical analysis can be determined.
- Two sera showed none Reactions when testing against membrane fraction II of the strain ORTH in the vertical analysis.
- a serum could nevertheless determined as positive by the horizontal analysis will.
- Fig. 12 the results of the two examples for the horizontal (HA) and the vertical analysis (VA) are shown side by side.
- the human sera, their SYNTYPs (syndrome type) and the phase assignment can be found in the left column in the same order as in FIGS. 4 and 8.
- the three following column groups are titled "VA", "HA” and "V / H”.
- the titles are respectively short forms of the v ertikalen A nalysis (VA), the h certainalen A nalysis (HA) or the combination of both forms of analysis (V / H).
- the column groups "VA” and "HA” have the same structure: The left column shows whether the serum in question was rated positive ("P") or negative ("-").
- the three subsequent columns show how many reactions the respective serum had with IgG antibodies (+), IgM antibodies ( ⁇ ) or if both an IgG and an IgM reaction were directed against the same antigen ( ⁇ ).
- the gap V / H shows the results of the combination of both types of analysis.
- Fig. 13 are the results of the three studies (CZR / Austria, Finland, Germany) comparative shown. All sera (control and patient groups), a total of 670, of the three studies were on the Vertical analysis rated. The sera of the control group, however the patient sera from Finland and Germany were also also classified via the HA. Both Only sera from the CZR / Austria study became patient sera Sera assessed via the HA, which in the VA as "negative" were found.
- FIG. 14 shows a direct comparison of the efficiency of VA and HA. It shows that on average about 72% of the sera rated as "positive" were recognized by both types of analysis. In addition, 19% of the sera were only detected by the HA and 9% of the sera only by the VA. In other words, only 91% of the sera would be detectable with the HA alone, and only 81% with the VA alone (always based on 100% through the combined application).
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DE19629543A DE19629543C2 (de) | 1996-07-22 | 1996-07-22 | Immunassay zum Nachweis von anti-B. burgdorferi Antikörpern und Verfahren zur Serodiagnose bei Lyme Borreliose, diagnostische Mittel und Testkits zur Durchführung der Verfahren |
DE19629543 | 1996-07-22 |
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EP0821237A2 true EP0821237A2 (fr) | 1998-01-28 |
EP0821237A3 EP0821237A3 (fr) | 1999-02-24 |
EP0821237B1 EP0821237B1 (fr) | 2004-03-03 |
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EP97112255A Expired - Lifetime EP0821237B1 (fr) | 1996-07-22 | 1997-07-17 | Essai immunologique pour la détection des anticorps anti-B. burgdorferi et méthode serodiagnostic pour Lyme borreliose, agent diagnostique et kits de tests pour la mise en oeuvre des procédés |
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EP (1) | EP0821237B1 (fr) |
AT (1) | ATE261123T1 (fr) |
DE (2) | DE19629543C2 (fr) |
DK (1) | DK0821237T3 (fr) |
Cited By (2)
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WO2001042790A2 (fr) * | 1999-12-08 | 2001-06-14 | Wita Gmbh | Agents et procedes pour le diagnostic de la borreliose de lyme et vaccin contre la borreliose |
EP1726960A1 (fr) * | 2005-05-23 | 2006-11-29 | Viramed Biotech AG | Moyens et procédés pour la détection des anticorps anti-Borrelia et kit pour utilisation dans le diagnostic de la borreliose de lyme |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
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WO1993004175A1 (fr) * | 1991-08-15 | 1993-03-04 | Smithkline Beecham Biologicals (S.A.) | Proteines osp a des sous-groupes borrelia burgdorferi, genes codeurs et vaccins |
WO1994025596A2 (fr) * | 1993-04-29 | 1994-11-10 | Immuno Aktiengesellschaft | FORMULATION IMMUNOGENE DE VACCINS A BASE D'ANTIGENES OspC, DESTINEE A LA PREVENTION ET AU TRAITEMENT DE LA MALADIE DE LYME ET PROCEDES DE RECOMBINAISON POUR LA PREPARATION DE CES ANTIGENES |
WO1995014781A2 (fr) * | 1993-11-24 | 1995-06-01 | The University Of Connecticut | Procedes de diagnostic precoce de la maladie de lyme |
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EP1016416A3 (fr) * | 1990-07-06 | 2002-10-23 | Wyeth | Vaccin contre la maladie de lyme et modèle pour l'évaluation de l'efficacité du vaccin |
US5547835A (en) * | 1993-01-07 | 1996-08-20 | Sequenom, Inc. | DNA sequencing by mass spectrometry |
-
1996
- 1996-07-22 DE DE19629543A patent/DE19629543C2/de not_active Expired - Fee Related
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Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
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WO1993004175A1 (fr) * | 1991-08-15 | 1993-03-04 | Smithkline Beecham Biologicals (S.A.) | Proteines osp a des sous-groupes borrelia burgdorferi, genes codeurs et vaccins |
WO1994025596A2 (fr) * | 1993-04-29 | 1994-11-10 | Immuno Aktiengesellschaft | FORMULATION IMMUNOGENE DE VACCINS A BASE D'ANTIGENES OspC, DESTINEE A LA PREVENTION ET AU TRAITEMENT DE LA MALADIE DE LYME ET PROCEDES DE RECOMBINAISON POUR LA PREPARATION DE CES ANTIGENES |
WO1995014781A2 (fr) * | 1993-11-24 | 1995-06-01 | The University Of Connecticut | Procedes de diagnostic precoce de la maladie de lyme |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001042790A2 (fr) * | 1999-12-08 | 2001-06-14 | Wita Gmbh | Agents et procedes pour le diagnostic de la borreliose de lyme et vaccin contre la borreliose |
WO2001042790A3 (fr) * | 1999-12-08 | 2001-12-06 | Wita Gmbh | Agents et procedes pour le diagnostic de la borreliose de lyme et vaccin contre la borreliose |
EP1726960A1 (fr) * | 2005-05-23 | 2006-11-29 | Viramed Biotech AG | Moyens et procédés pour la détection des anticorps anti-Borrelia et kit pour utilisation dans le diagnostic de la borreliose de lyme |
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Publication number | Publication date |
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ATE261123T1 (de) | 2004-03-15 |
DE19629543A1 (de) | 1998-01-29 |
EP0821237B1 (fr) | 2004-03-03 |
DK0821237T3 (da) | 2004-06-21 |
EP0821237A3 (fr) | 1999-02-24 |
DE59711357D1 (de) | 2004-04-08 |
DE19629543C2 (de) | 1999-02-11 |
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